The synthesis of organic molecules on solid-phase synthesis beads has experienced an explosion of interest since Merrifield's pioneering work in the peptide area several decades ago. In large part, this renaissance has been driven by the advent of combinatorial chemistry, which takes advantage of the ability to synthesize large and diverse libraries of compounds efficiently on solid support.
One inherent difficulty of producing large libraries by combinatorial chemistry is the problem of how to determine the reaction history in the form of the individual synthesis steps resulting in the synthesis of any given combinatorial library member. Without such information it is not possible to deconvolute the structure of the combinatorial library member.
When employing a large number of solid supports and a large number of synthesis steps and/or processing methods, the procedure of “deconvolution” is particularly difficult. In many practical cases, where high throughput screening and fast analysis is required, this problem is inherently associated with conventional methods for solid-phase synthesis.
Despite the tremendous practical advantages afforded by solid-phase synthesis, few reports have appeared in which a direct determination of the on-resin chemistry has been made possible in a practical way. Examples of techniques that have been used include radiography, nanoprobe nuclear magnetic resonance, single-bead fluorescence microscopy, IR spetroscopy, and optical analysis.
Combinatorial libraries may be assembled by a number of methods including the “split-and-recombine” methods described e.g. by Furka et al. (1988, 14th Int. Congr. Biochem., Prague, Czechoslovakia 5:47; 1991, Int. J. Pept. Protein Res. 37: 487-493) and by Lam et al. (1991, Nature 354: 82-84), and reviewed by Eichler et al. (1995, Medicinal Research Reviews 15 (6): 481-496) and by Balkenhohl et al. (1996, Angew. Chem. Int. Ed. Engl. 35: 2288-2337).
The split-and-recombine synthesis method involves dividing a plurality of solid supports such as polymer beads into n equal fractions representative of the number of available “building blocks” for each step of the synthesis (e.g., 20 L-amino acids, 4 different nucleotides etc.), coupling a single respective building block to each polymer bead of a corresponding fraction, and then thoroughly mixing the polymer beads of all the fractions together. This process is repeated for a total of x cycles to produce a stochastic collection of up to Nx different compounds.
The conventional split synthesis technologies referred to above present difficulties when it is desired to detect and isolate a combinatorial library member of interest. In this regard, it is necessary to first cleave the member from its solid support before identifying the member by techniques such as mass spectroscopy or HPLC. This is time consuming and cumbersome and in some cases, cleavage is not possible.
Janda (1994, Proc. Natl. Acad. Sci. USA 91: 10779-10785) describes a method in which each synthesis step of a combinatorial library member is followed by an independent coupling of an identifier tag to a solid support. Through a series of sequential chemical steps, a sequence of identifier tags are built up in parallel with the compounds being synthesised on the solid support. When the combinatorial synthesis is complete, the sequence of operations any particular solid support has gone through may be retraced by separately analysing the tag sequence. Accordingly, use of identifier tags in this manner provides a means whereby one can identify the building blocks sequentially added to an individual solid support during the synthesis of a member of a combinatorial library.
WO 98147838 discloses a method for the preparation of a chemical library on a plurality of synthesis particles comprising random features.
WO 93/06121 discloses a general stochastic method for synthesising a combinatorial compound library on solid supports from which library members may be cleaved to provide a soluble library. The identifier tag may be attached directly to a member of the library or to the solid support on which the member is synthesised. Tags such as oligonucleotides can be identified by sequencing or hybridisation. Amplification of oligonucleotide tags by PCR can be employed when only trace amounts of oligonucleotides are available for analysis. However, such identification methods are time consuming and inefficient.
U.S. Pat. No. 5,721,099 discloses a process for constructing complex combinatorial chemical libraries of compounds wherein each compound is produced by a single reaction series and is bound to an individual solid support on which is bound a combination of four distinguishable identifiers which differ from one another. The combination provides a specific formula comprising a tag component capable of analysis and a linking component capable of being selectively cleaved to release the tag component. Prior to analysis of a combinatorial library, each tag component must be cleaved from the support thus creating at least one additional step which is time consuming and inefficient.
Also, the above methods all rely on parallel, orthogonal synthesis of identifier tags which adds substantially to the time taken for completion of a combinatorial synthesis and has the potential of interfering with the synthesis.
Spectrometric encoding methods have also been described in which decoding of a library member is permitted by placing a solid support directly into a spectrometer for analysis. This eliminates the need for a chemical cleavage step. For example, Geysen et al. (1996, Chem. Biol. 3: 679-688) describe a method in which isotopically varied tags are used to encode a reaction history. A mass spectrometer is used to decode the reaction history by measuring the ratiometric signal afforded by the multiply isotopically labelled tags. A disadvantage of this method is the relatively small number of multiply isotopically labeled reagents that are commercially available.
Optical encoding techniques have also been described in which the absorption or fluorescence emission spectrum of a solid support is measured. Sebestyen et al. (1993, Pept. 1992 Proc. 22nd Eur. Pept. Symp. 63-64), Campian et al. (1994, In Innovation and Perspectives on Solid Phase Synthesis; Epton, R., Birmingham: Mayflower, 469-472), and Egner et al. (1997, Chem. Commun. 735-736) have described the use of both chromophoric and/or fluorescent tags for bead labeling in peptide combinatorial synthesis. Although this use provides an advantage for deconvoluting the structure of a library member by determining the absorption or fluorescence emission spectrum of a bead, the encoding of a large library would require the use of many chromophores or fluorophores where spectral superimposition would be a likely drawback.
WO 95/32425 discloses the coupling on beads of (i) fluorescently labelled tags having intensities that differ by a factor of at least 2, and/or (ii) multiple different fluorescent tags that can be used in varying ratios, to encode a combinatorial library. Such beads may be used in concert with flow cytometry to construct a series of combinatorial libraries by split synthesis procedure. Although this method has advantages in relation to providing a lead structure, it is necessary to construct and analyse multiple libraries commensurate with the number of stages used for the combinatorial synthesis, which is cumbersome and time consuming.
WO 97/15390 describes a physical encoding system in which chemically inert solid particles are each labelled with a unique machine readable code. The code may be a binary code although higher codes and alphanumerics are contemplated. The code may consist of surface deformations including pits, holes, hollows, grooves or notches or any combination of these. Such deformations are applied by micro-machining. Alternatively, the code may reside in the shape of the particle itself. Solid particles comprising a first phase for combinatorial synthesis and a second phase containing a machine readable code are exemplified wherein the second phase may be superimposed on, or encapsulated within, the first phase. The microscopic code on the particles may be interrogated and read using a microscope-based image capture and processing system. The machine readable code may be read “on-line” between different process steps of a combinatorial synthesis thus allowing the process sequence, or audit trail, for each bead to be recorded.
Nano bar coding for bioanalysis has also been described by Keating, Natan and co-workers (Science, 2001, vol. 294, 137).
Xu et al. (2003) Nucleic Acid Research 31(8):e43 describes the use of combinations of fluorescent semiconductor nanocrystals to encode microspheres. The nanocrystals are too small to allow visualisation of their spatial location in the bead. Furthermore, as overlap of emission spectra needs to be avoided, the number of different nanocrystals that can be used in one bead will be limited.
WO 00/32542 discloses high throughput screening based on carriers having distinctive codes such as electronmagnetic radiation-related compounds. Similar methods have been described by Battersby et al. (2001, Drug Discovery Today, vol. 6, no. 12 (Suppl.), S19-26); Battersby and Trau (2002, Trends in Biotechnology, vol. 20, no. 4, 167-173; Meza (2000, Drug Discovery Today, vo. 1, no. 1, 38-41), and by Farrer et al. (2002, J. Am. Chem. Soc., vol. 124, no. 9, p. 1994-2003).
Many of the disadvantages of the known methods described above, as well as many of the needs not met by these methods, are overcome by the present invention, which, as described herein below, provides several advantages over the above-described prior art methods.